Neuregulins (NRG) have emerged as key regulators of synaptic signalling. These transmembrane proteins are encoded by four genes (NRG-1, -2, -3 and -4), and their diversity is further increased by alternate RNA splicing and promoter usage and in particular by posttranslational modifications like proteolytic processing which leads to release of soluble isoforms from membrane-bound holoproteins. Moreover there is evidence of phosphorylation and glycosylation (Buonanno and Fischbach 2001). They are characterized by different extracellular domains and are ligands of ErbB receptor tyrosine kinases, which have downstream connotations to neuroinflammation and gene transcription (Holbro and Hynes 2004). In particular, soluble isoforms of NRG-1 are produced from the transmembrane form of NRG through proteolytic cleavage during electrical stimulation and subsequently secreted as activity-dependent synaptic modulators (Ozaki et al. 2004).
A truncated isoform of NRG-1, presumably β1, comprising the N-terminal extracellular domain (ECD) of the entire membrane protein, which has been found to be correlated to learning and memory (Schillo et al. 2005a; WO03/014156). Functional studies have demonstrated, that NRG-1 directly regulates NMDA receptor subunit composition (Ozaki et al. 1997; Eilam et al. 1998). Moreover it has been shown that NRG-1 fragments of this type have neuroprotective properties in vivo by antiapoptotic effects (Xu et al. 2005A, Xu et al. 2005B, Xu et al. 2004).
Very recently it became clear that NRG-1 has a central role in human neurological diseases due to NRG-dependent regulation of NMDA receptors (Schrattenholz and Soskic 2006), and subsequent downstream events like excitotoxicity, neuroinflammation and apoptosis (see FIG. 1 for summary). There are results showing that NRG 1 plays a pivotal role in conditions ranging from amyotrophic lateral sclerosis, Alzheimer's and Parkinson′ disease, to stroke and schizophrenia (Britsch 2007).
This fundamental significance of NRG-1 implies that next to neuroprotection and a positive role in cognition-related learning and memory, NRG-1 represents a crucial neurotrophic factor in regeneration of neuronal tissue after a variety of lesions, in a variety of specific brain regions and cell types. Obviously it is the crucial factor for maintenance and repair of the integrity of neuronal circuitry: neuroprotective and with roles in correct regeneration after loss of function, as well as in the formation of activity-dependent neuronal plasticity.
The interest in Neuregulin 1 β was further fueled considerably when Kastin et al., 2004, showed that Neuregulin 1 β is able to cross the blood-brain barrier. That opened the perspective for the therapeutic usage of Neuregulin 1 β.
Latest research proved the breadth of application in neuroprotection. Independently it was shown in two publications that Neuregulin 1 is also a substrate of BACE (β-secretase, β-amyloid converting enzyme), which indicates the relevance of Neuregulin 1 in Alzheimer's disease (Glabe 2006; Schubert 2006).
Further, it was found that in Schwann cells neuregulin-1 increases the transcription of the 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase, the rate-limiting enzyme for cholesterol biosynthesis in Schwann cells (Pertusa et al. 2007). This has far reaching implications for all conditions where the myelin sheath is affected, e.g. schizophrenia and multiple sclerosis, or cognition-related functions, where so-called “cholesterol-rich rafts” are involved (Schrattenholz and Soskic 2006). Schwann cell surrounding axons express NRG1 receptors ErbB2/ErbB3 and soluble NRG1α and β under physiological conditions Following denervation, adult Schwann cells leave the contact with axon, change their morphology, stop expressing NRG1β, and upregulate NRG1α and ErbE32/ErbB3 expression (Geuna et al, 2007; Karoutzou et al. 2007).
In addition, genetic epidemiologic research shows the clear association of Neuregulin 1 to schizophrenia and to Alzheimer disease, and in particular to its psychotic forms (Farmer et al., 2007).
Some recent genetic population analyses show, that certain NRG-1-SNP's are associated with Alzheimer and schizophrenia (Go et al. 2005; Scolnick et al. 2006; Ross et al. 2006; Meeks et al. 2006; Farmer et al. 2007). The implications of these findings are related to other proteins of the functional NRG-containing complex depicted in FIG. 1 (ErbB receptor: (Benzel et al. 2007; Thomson et al. 2007; Hahn et al. 2006). There is also an implication for NRG-1 in multiple sclerosis (Esper et al. 2006).
There are results suggesting that the molecular mechanism of the association between NRG1 risk alleles and schizophrenia may include down-regulation of nicotinic acetylcholine receptors of alpha7subtype (Mathew et al. 2007).
According to the present invention it was found that recombinant soluble Neuregulin-1 β isoforms show pharmaceutical efficacy in animal models for learning and memory, schizophrenia, Alzheimer's disease and Parkinson's disease. After i.v. administration, Neuregulin-1 β isoforms were active at concentrations which are significantly lower than concentrations of control medicaments.
Thus, a first aspect of the present invention is the use of a recombinant soluble Neuregulin-1 isoform for the manufacture of a medicament for the treatment of neurological conditions, particularly of cognition-related neurological conditions.
A further aspect of the present invention is a pharmaceutical composition or kit comprising (i) a recombinant soluble Neuregulin-1 isoform and (ii) a further medicament particularly for the treatment of neurological conditions, particularly of cognition-related neurological conditions.
Still a further aspect of the present invention is the use of a recombinant soluble Neuregulin-1 isoform for memory and cognition enhancement for the manufacture of a medicament.
Still a further aspect of the present invention is a method of treating a neurological condition comprising administering a recombinant soluble Neuregulin-1 isoform in a pharmaceutically effective amount to a subject in need thereof.
Still a further aspect of the present invention is a method for enhancing memory and cognition comprising administering a recombinant soluble Neuregulin-1 isoform in a pharmaceutically effective amount to a subject in need thereof.
Still a further aspect of the present invention is a co-administration of a recombinant soluble Neuregulin-1 isoform together with a further medicament.
According to the present invention, soluble Neuregulin-1 isoforms have been found to be effective for the treatment of neurological conditions, particularly conditions, such as psychotic disorders like schizophrenia, bipolar disorder and depression, neurodegenerative disorders, like Parkinson's disease, Alzheimer's disease, Multiple Sclerosis (MS), or Amylotrophic Lateral Sclerosis (ALS), epilepsy or neurological injury like stroke, traumatic brain injury and spinal chord injury. Preferred is the treatment of schizophrenia, in particular cognition-related aspects of schizophrenia, Parkinson's disease and Alzheimer's disease. Further, the invention also refers to the use of recombinant soluble Neuregulin-1 isoforms for memory and cognition enhancement, particularly for reducing and/or inhibiting memory and cognition loss associated with a neurological condition such as Alzheimer's disease and schizophrenia.
The recombinant soluble Neuregulin-1 isoform is preferably a human Neuregulin-1 isoform, i.e. a recombinant isoform comprising the primary amino acid sequence of a naturally occurring human Neuregulin-1 isoform or a sequence which has a identity of at least 90%, preferably at least 95% and most preferably of at least 98% based on the total length of the recombinant isoform.
The soluble recombinant Neuregulin-1 isoform of the present invention preferably comprises at least a portion of the extracellular domain of the corresponding Neuregulin-1, e.g. at least a portion of the extracellular domain of a human Neuregulin, e.g. human Neuregulin-1 β.
The recombinant soluble Neuregulin isoform of the present invention preferably has a length of up to 250 amino acids, e.g. 150 to 250 amino acids. The molecular weight of the Neuregulin isoform is preferably of about 15 to about 35 KD, particularly about 25 to about 32 KD, as measured e.g. by SDS-polyacrylamide electrophoresis (PAGE). The recombinant soluble Neuregulin-1 isoform, particularly the recombinant Neuregulin-1 β isoform, has an isoelectric point (pI) of about 4 to about 9.5, preferably of about 4 to about 6. The isoform may be an unmodified polypeptide which consists of an unmodified amino acid sequence or a modified polypeptide, wherein the modification may be selected from phosphorylation, glycosylation, methylation, myristylation, oxidation and any combination thereof. In an especially preferred embodiment, the Neuregulin-1 isoform comprises at least one phosphorylated amino acid residue. Further, the present invention encompasses conjugation to heterologous moieties such as poly(alkyleneoxide) moieties, particularly polyethylene glycol moieties.
The recombinant soluble isoforms may be administered according to any route by which effective delivery into the target tissue, e.g. the nervous system, particularly the central nervous system, such as brain and/or spinal chord, is achieved. It was found that pharmaceutically effective concentrations of Neuregulin isoforms may be achieved by systemic administration. For example, the isoforms may be administered by injection or infusion, e.g. by intravenous injection. The isoforms are preferably administered in an amount of 0.1 to 5000 ng/kg body weight, particularly in an amount of 2 to 1000 ng/kg body weight and more particularly in an amount of 3 to 600 ng/kg body weight of the subject to be treated, depending on the type and severity of the condition to be treated. In other embodiments of the present invention the soluble isoforms may also be administered locally, e.g. by direct administration into the central nervous system, e.g. into the spinal chord and/or into the brain. Also administration at higher dosages of up to 500 μg/kg by i.p. or s.c. injections, or inhalation devices are may be considered. Preferably the subject to be treated is a mammal, more preferably a human patient.
The soluble recombinant Neuregulin-1 isoforms may be administered as a stand-alone medication, i.e. as a monotherapy or as a co-medication, i.e. in combination with a further medicament, particularly with a further medicament which is suitable for the treatment of a neurological condition. Examples of further medicaments are compounds affecting catecholamine metabolism, acetylcholine esterase inhibitors, MAO-B- or COMT-inhibitors, Memantine-type channel blockers, dopamine or serotonine receptor agonists or antagonists, catecholamine or serotonine reuptake inhibitors or any type of antipsychotic medicaments like clozapine or olanzapine or gabapentin-like drugs, particularly in the treatment of Alzheimer's and Parkinson's diseases, schizophrenia, bipolar disorder, depression or other neurological conditions. Additional examples of further medicaments are neuroprotective agents such as PARP-1 inhibitors, e.g. as disclosed in WO 2006/008118 and WO 2006/008119, which are herein incorporated by reference.
Thus, an embodiment of the present invention refers to the combination of a recombinant soluble Neuregulin-1 isoform as described herein with a medicament for the treatment of psychotic disorders such as schizophrenia, bipolar disorders and depression, e.g. olanzapine or clozapine. A further embodiment refers to the combination of a recombinant soluble Neuregulin-1 isoform and a medicament for the treatment of a neurodegenerative disease such as Parkinson's disease, Alzheimer's disease, MS or ALS. Still a further embodiment refers to the combination of a recombinant soluble Neuregulin-1 isoform and a medicament for the treatment of neurological injury, such as stroke, traumatic brain injury or spinal chord injury.
The combination therapy may be effected by co-administering the recombinant soluble Neuregulin-1 isoform and the further medicament in the form of a pharmaceutical composition or kit, wherein the individual medicaments are administered by separate or common administration.
The Neuregulin-1 isoform may be a Neuregulin-1 Type I, Type II, Type III, Type IV, Type V or Type VI isoform, preferably a Neuregulin-1 β isoform, a Neuregulin-1 α isoform or a Sensory and motor neuron-derived factor (SMDF) isoform, particularly a Neuregulin-1 β isoform and more particularly a human Neuregulin-1 β isoform.
Neuregulin 1 β isoforms are actively transported through the blood brain barrier. The excellent bioavailability of Neuregulin 1 β in the brain after i.v./i.p. Injection, as shown in the Examples paves the way towards a therapeutic application of NRG 1 β.
Its combination of antiapoptotic, myelin-stabilizing, anti-inflammatory properties, together with the direct interaction with BACE opens opportunities in the treatment of stroke, Alzheimer, MS and schizophrenia and other neurological conditions.
As outlined above, the present application encompasses the use of unmodified and modified Neuregulin-1 isoforms, particularly Neuregulin-1 β isoforms. There is evidence that posttranslational modifications like proteolytic processing, phosphorylation and glycosylation take place at certain amino acid residues of the Neuregulin-1, and in particular its extracellular domain. In particular the release of soluble fragments of Neuregulin-1 has been reported (Buonanno and Fischbach 2001; Fischbach 2007). Potential oxidation has been reported as well (Nadri et al. 2007).
The present inventors have obtained evidence that preferred physiologically active Neuregulin-1 β isoforms comprise the extracellular domain of Neuregulin-1 β or a part thereof which has been post-translationally modified. Preferably, the isoforms have been modified by phosphorylation, wherein 1, 2, 3 or more amino acid side chain residues, particularly side chain residues having an OH-group such as Tyr, Ser or Thr, have been phosphorylated. Preferred phosphorylation sites are located at amino acid positions 79-82, 133-136 and/or 158-161 (nomenclature according to Falquet et al., 2002). Further preferred phosphorylation sites are located at amino acids 12-14, 30-32 and/or 85-87. Further potential modification sites are amidation sites, preferably located at positions 22-25 and/or 30-33, glycosylation sites at positions 150-153, 156-159 and/or 204-207, and myristylation sites, preferably located at positions 94-99, 149-154, 168-173, 175-180 and/or 202-207 according to the nomenclature of Falquet et al. 2002.
In the following, the relevance of the experimental data according to the present application are explained with regard to preferred medical indications.
Schizophrenia
Schizophrenia is a serious and disabling mental disorder with symptoms such as auditory hallucinations, disordered thinking and delusions, avolition, anhedonia, blunted affect and apathy. Epidemiological, clinical, neuropsychological, and neurophysiological studies have provided substantial evidence that abnormalities in brain development and ongoing neuroplasticity play important roles in the pathogenesis of the disorder (Arnold et al. 2005).
Schizophrenia is thought to include a disorder of dopaminergic neurotransmission, but modulation of the dopaminergic system by glutamatergic neurotransmission seems to play a key role. This view is supported by genetic findings of the neuregulin- and dysbindin genes, which have functional impact on the glutamatergic system (Muller and Schwarz 2006). What has become increasingly clear is that several regions that are likely to contain genes (including neuregulins) contributing to schizophrenia are also relevant to bipolar affective disorder, a finding supported by recent twin data (Farmer et al. 2007; Owen et al. 2007).
Neuregulin-1, which is a psychosis susceptibility gene with effects on neuronal migration, axon guidance and myelination that could potentially explain findings of abnormal anatomical and functional connectivity in schizophrenia and bipolar disorder (McIntosh et al. 2007).
There is an ever increasing body of evidence of a genetic linkage of Neuregulin 1 to schizophrenia (review: Farmer et al., 2007). The enhancement of glutamate, GABA and nicotinic neurotransmission by Neuregulin-1 (Fischbach 2007; Woo et al. 2007; Li et al. 2007) is relevant in this context, as well as implication with brain inflammation (Hanninen et al. 2007).
The regulation of 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase, the rate-limiting enzyme for cholesterol biosynthesis (Pertusa et al. 2007), important for myelinisation, is assumed to have implications in this condition as well.
The fact that among genetic risk factors common to schizophrenia, bipolar disorder and depression, NRG1 plays an outstanding role, has triggered suggestions that genes implicated in these psychoses such as NRG-1 may eventually provide the basis for classification based on biology rather than symptoms, and lead to novel treatment strategies for these complex brain disorders (Blackwood et al. 2007; Bertram et al. 2007).
The experimental data of the present application demonstrate the effectiveness of administration of a soluble recombinant Neuregulin-1 β isoform in an experimental model of schizophrenia.
Alzheimer's Disease
Initial research by the inventors showed that Neuregulin 1 β is diminished in post mortem sections of hippocampi of brains of Alzheimer's patients as compared to age-matched controls (Sommer et al., 2004) with a clear positive correlation of the soluble fragment of Neuregulin-1 with learning performance in a radial maze test (Sommer et al., 2004).
There are numerous reports demonstrating the role of NRG-1 in activity-dependent synaptic changes (Xie et al. 2006; Kwon et al. 2005; Rimer et al. 2005; Bao et al. 2004; Yang et al. 2005) important for learning and memory (Ozaki et al. 1997; Ozaki et al. 2004; Golub et al. 2004; Schillo et al. 2005b). As shown below, the NRG1β fragment containing the extracellular domain was clearly associated with learning in a behavioural animal model. Showing decreased expression of the protein in post mortem brain slices of the hippocampal regions (responsible for short term memory formation) of Alzheimer patients as compared to age-matched controls could demonstrate the absence of memory-related synaptic activity, in regions of apparently still healthy neurons.
Very recent discoveries (Hu et al. 2006; Glabe 2006; Schubert 2006) show that NRG 1 is processed by BACE1 (=β secretase), an enzyme that helps generate clumps of amyloid-β in the brains of people with Alzheimer disease, which explains the link to Alzheimer's disease, its concomitant role in myelin formation relates to the neurotrophic properties of NRG 1 (Hu et al., 2006; Glabe 2006; Schubert 2006). The enzyme, BACE1 (beta-site amyloid precursor protein—cleaving enzyme 1), is required to cleave amyloid-β from a larger precursor. (After BACE1-mediated cleavage, the presenilin-containing complex γ-secretase makes the final cleavage, liberating amyloid-β.
The cleavage of NRG by secretases is crucial for nerve myelination. Just like amyloid precursor protein, neuregulin 1 is also cleaved by β-secretase. Proteolytic cleavage of neuregulin 1 by β-secretase is critical for peripheral nerve myelination by Schwann cells. Drugs that target β-secretase could affect peripheral nerve development and function.
The in initial observation was by the group of Haass (Willem et al. 2006), who found that BACE1 seems also to be required for myelination. Peripheral nerve myelination occurs early in life, so it is unclear how BACE1 inhibition might affect older animals. There are indications that BACE1 also has a role in myelination of the central nervous system. Transgenic animals deficient in BACE-1 had myelin defects in the peripheral nerves
Also in the context of neurodegeneration and Alzheimer's disease, the recent discovery of enhancement of glutamate, GABA and nicotinic neurotransmission by Neuregulin-1 (Fischbach 2007; Woo et al. 2007; Li et al. 2007) is relevant.
The experimental data of the present application demonstrate the effectiveness of administration of a soluble recombinant Neuregulin-1 β isoform in an experimental model of Alzheimer's disease.
Stroke, Traumatic Brain Injury
A series of stroke-related in vivo experiments by independent external research in the US, demonstrate neuroprotection by Neuregulin 1 which by itself is antiapoptotic (Xu et al., 2004, 2005 and 2006; Guo et al., 2006)
NRG-1 reduces neuronal damage and improves neurological outcome after middle cerebral artery occlusion (a common stroke model) (Xu et al. 2005b; Xu et al. 2004; Xu et al. 2006; Guo et al. 2006).
In the same study about the therapeutic efficacy and mechanism of recombinant human NRG-1 in attenuating brain injury by ischemia/reperfusion, it was found that NRG is antiapoptotic. NRG-1 (3.0 ng/kg) was applied intravascularly 10 min before middle cerebral artery occlusion (MCAO) and subsequent focal cerebral ischemia for 90 min and reperfusion for 24 h.
The data of the present invention demonstrate that administration of recombinant soluble Neuregulin-1 isoforms at low concentration has a significant pharmacological effect and thus is assumed to be effective in models of stroke and traumatic brain injury.
In the following, the present application is explained in more detail by the Figures and Examples given herein below.